Structural Analysis of Testicular Appendices in Patients with Cryptorchidism ______Guilherme D

ORIGINAL Article Vol. 39 (2): 240-247, March - April, 2013 doi: 10.1590/S1677-5538.IBJU.2013.02.13

Structural analysis of testicular appendices in patients with cryptorchidism ______Guilherme D. Tostes, Suelen F. Costa, João P. de Carvalho, Waldemar S. Costa, Francisco J.B. Sampaio, Luciano A. Favorito

Urogenital Research Unit, Rio de Janeiro State University, Rio de Janeiro, Brazil

ABSTRACT ARTICLE INFO ______

Objectives: Report the incidence and structure of testicular appendices (TAs) in patients Key words: with cryptorchidism, comparing their incidence with epididymal anomalies (EA) and pa- Cryptorchidism; anatomy tency of the vaginal process (PVP) and analyzes the structure of TAs. and histology; Epididymis; Material and Methods: We studied 72 testes of patients with cryptorchidism (average of abnormalities 6 years), and 8 testes from patients with hydroceles (average of 9 years). We analyzed the relations among the testis, epididymis and PVP and prevalence and histology of the TAs. Int Braz J Urol. 2013; 39: 240-7 The appendices of 10 patients with cryptorchidism and 8 with hydrocele were dissected ______and embedded in paraffin and stained with Masson trichrome; Weigert and Picro-Sirius Red with polarization and immunohistochemistry analysis of the collagen type III fibers Submitted for publication: to observe collagen. The stereological analysis was done with the software Image Pro October 03, 2012 and Image J, using a grid to determine volumetric densities (Vv). Means were statistically ______compared using the ANOVA and unpaired T test (p < 0.05). Results: Of the 72 testes with cryptorchidism, 20 (27.77%) presented EA, 41 (56.9%) Accepted after revision: had PVP and 44 (61.1%) had TAs. Of the 44 testes with cryptorchidism and appendices, January 10, 2013 30 (68.18%) presented PVP and 11 (25%) presented EA. There was no alteration of the epithelium in the appendices of patients in both groups. Stereological analysis documented the prevalence of ESFs (mean of 1.48%), prevalence of veins (mean of 10.11%) and decrease (p = 0.14) of SMCs in the TAs of patients with cryptorchidism (mean = 4.93%). Collagen III prevailed in the TAs of patients with cryptorchidism. Conclusion: The testicular appendices presented significant structural alteration in the patients with cryptorchidism, indicating that TAs present a structural remodeling.

INTRODUCTION the most frequently twisted of the four testicular appendages (3-5). The other are the appendix of Testicular and epididymal appendices have the epididymis, which is a remnant of the wolffian been considered congenital anomalies (1). The duct, the paradidymis and the vas aberrant. These appendix testis or hydatid of Morgagni is believed vestigial structures have a similar histology, com- to be the embryologic remnant of the cranial end posed of gelatinous and vascular connective tissue of the Mullerian or paramesonephric duct (2). It covered with a columnar epithelium (6). is present in more than 90% of males, it varies in The functions of testicular appendices are size from 1-10 mm in diameter and it is usually controversial, but they may control the amount pedunculated, which predisposes to torsion. It is of serous fluid within the space of the vaginal

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process (7). Another hypothesis suggests that the and epididymis; and Type VI - epididymal atresia. surface epithelium, subepithelial capillaries and Type I and II relations are considered normal whi- lymphatic vessels of testicular appendices form a le the other types are considered to be anatomical functional unit (8). Some studies have analyzed anomalies (12). To analyze the vaginal process we the embryology (9) and structure of the testicular considered two situations: (a) complete oblitera- and epididymal appendices (3,10). tion of the vaginal process between the internal Studies of the incidence of these structures inguinal ring and the superior pole of the testis; in patients with cryptorchidism and comparison and (b) complete patency of the vaginal process. with individuals without testicular position ano- In relation to the testicular appendices, malies are rare in the literature (11). There are no we analyzed the following situations in the two published studies analyzing the structure of testi- groups during the surgeries: I) absence of testicular appendices in patients with cryptorchidism. cular and epididymal appendices; II) presence of This work reports the incidence and struc- testicular appendix only; III) presence of epidi- ture of testicular appendices (TAs) in patients with dymal appendix only; IV) presence of testicu- cryptorchidism, comparing their incidence with lar and epididymal appendices; V) presence of epididymal anomalies and patency of the vaginal two epididymal appendices and one testicular process and analyzes the structure of testicular appendix; and VI) presence of paradidymis or appendices to assess whether their architecture is vas aberrans of Haller. altered when cryptorchidism is present. The appendices of 10 patients with cryptorchidism and 8 patients with hydrocele were MATERIALS AND METHODS separated from the other structures and fixed in 10% buffered formalin, and routinely processed The present work received institutional re- for paraffin embedding. Then 5 µm thick sections view committee and parent approval and has been were obtained at 200 µm- intervals. Smooth mus- carried out in accordance to the ethical standards cle cells, connective tissue and elastic system fi- of the responsible institutional committee on hu- bers were studied by histochemical and immuno- man experimentation. labeling methods. We analyzed 72 testes from 55 patients The sections were stained with hemato- with cryptorchidism (17 with bilateral cryptorchi- xylin-eosin to assess the integrity of the tissue. dism) and compared them with a control group We performed the following stainings: Masson’s of 8 testes from patients with hydroceles. The trichrome, in order to quantify connective tissue patients with cryptorchidism had ages from 1 to and smooth muscle, and Weigert resorcin fucsin 13 years (average of 6 years), while those in the with previous oxidation in order to observe elas- control group ranged from 1 to 14 years old (ave- tic system fibers. Connective tissue, smooth mus- rage of 9 years). In the two groups, we analyzed cle cells and elastic system fibers were quantified the relations among the testis, epididymis and pa- by a digital method (14). Five sections per speci- tency of the vaginal process and the incidence and men were stained, and five fields of each section histology of the testicular appendices. were selected. All selected fields were photogra- To analyze the relations between the testis phed with an Olympus DP70 camera coupled to and epididymis during surgery, we used a previous an Olympus BX51 microscope. The images were classification (12,13). By this classification, there processed and saved with the Image Pro software. are six types of relations between the testis and The tissue was quantified using the Image J sof- epididymis: Type I - epididymis attached to the tware to determine the area density (Ad) of each testis at the head and tail; Type II - epididymis component (14). Immunolabeling was performed totally attached to the testis; Type III - epididymis to confirm the results. For smooth muscle cells attached to the testis only at the head; Type IV - (SMCs), Monoclonal Alfa Actin Antibody 08-0106 epididymis attached to the testis only at the tail; (Zymed Laboratories, San Francisco, CA, United Type V - no visible connection between the testis States) was used. To confirm the results for elastic

241 ibju | Analysis of testicular appendices in cryptorchidism

fibers (EF), Monoclonal Elastin Antibody ab 9519 (87.5%), 1 (14.28%) had epididymal anomaly and (Abcam Laboratories, Cambridge, MA, United Sta- 1 (14.28%) had patent vaginal process. tes) was used. Of the 44 testes with cryptorchidism and Means were statistically compared using appendices, 30 (68.18%) presented patent vaginal the unpaired t-test and linear regression was per- process and 11 (25%) showed epididymal ano- formed when applicable. Statistical relevance was malies. Of the 28 testes with cryptorchidism and accepted if p < 0.05. without appendices, 11 (39.28%) presented patent vaginal process and 9 (32.14%) showed epididymal RESULTS anomalies. The relation between the type of testicular appendix found and the patency of the vaginal Of the 72 testes with cryptorchidism, 7 process and the presence of epididymal anoma- were abdominal (9.7%), 54 (75%) were located in lies is shown in Table-2. There was no statistically the inguinal canal and 11 (15.3%) were supra-scro- significant difference in relation to the patency of tal. Of the testis with cryptorchidism, 20 (27.77%) vaginal process between the patients with cryptor- presented epididymal anomalies, 41 (56.9%) had chidism and appendices with the control group (p patent vaginal process and 44 (61.1%) had testi- = 0.053) and with the patients with cryptorchidism cular appendices. The prevalence of the testicular without appendices (p = 0.252). Also there was no appendices in the testis with cryptorchidism and statistically significant difference in relation to the their relation with the testicular position are re- epididimal anomalies between the patients with ported in Table-1. There was no statistically signi- cryptorchidism and appendices with the control ficant difference in relation to the location of the group (p = 0.305) and with the patients with cryp- appendices (p = 0.0652). torchidism without appendices (p = 0.6830). Of the Of the 7 abdominal testes, all had patent 55 patients, 17 had bilateral cryptorchidism, for a vaginal process and 4 (57%) had testicular appen- total of 34 testes. Of these, 1 was abdominal, 24 dices. Of the 54 testes located in the canal, 31 were located in the canal and 9 were supra-scrotal. (57%) had appendices, and of these, 21 (38.8%) Of the 34 testes of this group, 25 (73.5%) had testi- presented patent vaginal process and 19 (35.8%) cular appendices, with 13 being right testes and 12 had epididymal anomalies. Of the 11 cases of left ones, with no statistical difference. supra-scrotal testes, 9 (82%) had appendices, 5 Sections stained with Masson’s trichrome (55.5%) had patency of the vaginal process and 1 from the testicular appendix demonstrated a vascu- (11.1%) presented epididymal anomaly. Finally, of lar stroma lined with pseudocolumnar epithelium. the 8 cases in the control group, 7 had appendices The stroma consisted of loose connective tissue

Table 1 – Incidence of the testicular and epididymal appendices studied with respect to position of the testes with cryptorchidism and the testes in the control group. There was no statistically significant difference in relation to the location of the appendices (p = 0.0652).

Appendices Abdomen Canal Supra-Scrotal Control

No appendix 3 23 2 1

Testicular appendix 1 23 7 5

Epididymal appendix 2 4 0 1

Testicular and epididymal appendix 1 4 2 1

Total 7 54 11 8

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Table 2 – Relation between the type of appendix found and the patency of the vaginal process and presence of epididymal anomaly. There was no statistically significant difference in relation to the patency of vaginal process (p = 0.252) and epididymal anomalies (p = 0.6830) between the patients with cryptorchidism and appendices with the patients with cryptorchidism without appendices.

Appendices Patent vaginal process Epididymal anomaly

Testicular (31) 20 5

Epididymal (6) 5 4

Testicular and Epididymal (7) 5 2

Total (44-100%) 30 (68.18%) 11(25%) containing blood vessels, fibroblasts and varying lined with a pseudostratified columnar epithelium numbers of acini (gland-like structures), which (Figure-1B). There was no apparent alteration be- were lined with a non-ciliated columnar epithe- tween the epithelium in the appendices of patients lium (Figure-1A). The epididymal appendices were with cryptorchidism and in the control group, as vesicular structures. The cavity of the vesicle was seen in Figures 1A and 1C.

Figure 1 - Appendix epithelium. A) Patient with cryptorchidism with 3-years-old, showing the vascular stroma lined with pseudostratified epithelium of the testicular appendix. Masson’s trichrome x200. B) Patient of the control group with 13-year-old, showing epididymal appendix with cavitation covered by pseudostratified epithelium. Masson’s trichrome x 200. C) Patient of the control group with 13-years-old, showing pseudostratified epithelium of the entire testicular appendix. Masson’s trichrome x200.

A B

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Stereological analysis documented a pre- ces of the patients with cryptorchidism (mean of valence of elastic fibers in the testicular appen- 10.11%, standard deviation of 6.88) in relation to dices of the patients with cryptorchidism (mean the control group (mean of 4.77%, standard devia- of 1.48%) in comparison to the appendices of the tion of 1.9). This difference was statistically sig- patients with a hydrocele testis (mean of 0.29%), nificant (p < 0.001). Figure-3 shows the testicular a result that was not statistically different (p = appendices in patients with cryptorchidism and 0.22). Figure-2 shows the testicular appendices in those of the control group, stained by Masson’s patients with cryptorchidism and in the control trichrome. A greater number of vessels can be ob- group stained by Weigert’s resorcin-fucsin with served in the testes of patients with cryptorchi- oxidation. The testes of the patients with cryptor- dism. chidism contain a larger quantity of elastic fibers. Stereology analysis showed a decrease wi- Stereological analysis also showed a grea- thout significance (p = 0.14) of smooth muscle ter incidence of vessels in the testicular appendi- cells in the appendices of patients with cryptor-

Figure 2 - Elastic system fibers of the testicular appendix. A) Testicular appendix of 13-year-old patient with cryptorchidism. Weigert x400. B) Testicular appendix of 13-year-old patient of the control group. Weigert x400. Note the marked increase of elastic fibers (brown) in the patient with cryptorchidism when compared to the control.

A B

Figure 3 - Testicular appendix vessels. A) Testicular appendix of 2 years-old patient with cryptorchidism. Masson’s trichrome X200. B) Testicular appendix of 1 year-old patient in the control group. Masson’s trichrome X200. Note the marked increase of vessels in the appendix of the cryptorchidic patient.

A B

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chidism (mean = 4.93%) in comparison with the ned with Picro Sirius Red with polarization. The control group (mean = 9.032%). Figure-4 contains photomicrographs presented a large difference in photomicrographs comparing the smooth muscle colors between the groups. This difference can su- arrangement in the cryptorchidism and the con- ggest changes in the collagen fiber organization. trol group appendices. In the qualitative analysis, type III colla- DISCUSSION gen was observed in both groups, although type III collagen was prevalent in the testicular appen- Knowledge of the presence, form and loca- dices of patients with cryptorchidism and type I tion of testicular and epididymal appendices is im- was prevalent in the patients of the control group. portant in clinical practice because of the possibility Figure-5 shows examples of the two groups stai- of torsion of these structures, their association with

Figure 4 – Distribution of smooth muscle cells of the testicular appendix. A) Testicular appendix of 3-year-old patient with cryptorchidism. Imunohistochemical staining for alfha-actin X200. B) Testicular appendix of 11-year-old patient of the control group. Imunohistochemical staining for alfha-actin X200. Note the marked increase of SMCs in the appendix of the control patient when compared to the cryptorchidic patient.

A B

Figure 5 – Qualitative collagen distribution in testicular appendices. A) Testicular appendix of 3-year-old patient with cryptorchidism showing prevalence of yellowish color. Picrosirius-polarization method, X400. B) Testicular appendix of 11-years- old patient of the control group showing prevalence of greenish color. Picrosirius- polarization method, X400.

A B

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anatomical anomalies and the higher incidence of Reports of structural and ultrastructural tumors (3,15). Cryptorchidism is one of the most changes in the testicular appendices of patients common congenital anomalies among males, with with cryptorchidism are rare (10,19). In this study a rate between 2 and 5% of full-term births, a rate we did not observe any alterations in the epithe- that can reach 30% in premature babies (16-18). lium of the testicular and epididymal appendices There are few studies in the literature on the in- between the patients with cryptorchidism and tho- cidence and analysis of the structure of testicular se in the control group, where the majority of ca- appendices in patients with cryptorchidism and ses were made up of patients with hydroceles. We their relation with epididymal anomalies and pa- found a large quantity of veins and connective tency of the vaginal process (11). tissue in both the testicular and epididymal appen- Previous studies have shown that testicular dices. These findings are in agreement with those appendices occurs significantly less often in pa- of Sahni et al. (10) and can confirm the theory that tients with cryptorchidism. This can indicate a pos- the epithelial layer of the appendices along with sible role of testicular appendices in the testicular the subepithelial capillaries and lymphatic vessels migration process (19). In the present study, 61.1% form a functional unit (8,19). of the testes with cryptorchidism presented testicu- In our samples, we observed the presence of lar appendices, a much higher rate than the 24% elastic fibers in the appendices, more so in those of reported by Jozsa (19). We did not find a significant patients with cryptorchidism. The increased synthe- difference in the number of appendices in the tes- sis of elastic fibers may be associated with excessive tis with cryptorchidism in relation to those of the distension of an organ (24). We also observed a de- control group, and also did not find a significant crease in the quantity of smooth muscle fibers and difference in the incidence of appendices in relation predominance of type III collagen in the patients to the testicular position in the patients with cryp- with cryptorchidism. We can speculate that the torchidism. smooth muscle fiber reduction is the primary event Cryptorchidism can be associated with va- in fibrotic tissue formation. This event is probably rious anatomical anomalies, but epididymal anoma- correlated to a hydrodistension process. It has been lies and patency of the vaginal process are among variously shown that a shift towards greenish color the most frequent (20-22). Epididymal anomalies in the Picrosirius polarization method is associated are associated with cryptorchidism, with prevalence with less organized and/or degraded collagen (25). that ranges from 36 to 79% (20,21). In this study we This color change can also occur in earlier phases found 27.7% of the testes with cryptorchidism also of the remodeling and repair of connective tissues, had epididymal anomalies and 56.9% had patency when the synthesis of type III collagen is enhanced. of the vaginal process. Of the 44 testes with appen- Thus, based on these findings, the results of the Pi- dices, 68.18% had patency of the vaginal process crosirius-polarization method suggest that collagen and 25% presented epididymal anomalies. There matrix at the testicular appendices in patients with was no significant difference between the incidence cryptorchidism is disrupted or degraded, rather than of epididymal anomalies or patency of the vaginal fibrotic, which is consistent with higher hydrostatic process in the patients with cryptorchidism with pressure. These findings lead us to suggest that in and without appendices. testes without complete migration, testicular appen- Previous studies have shown that the tes- dices may result from the higher intra- abdominal ticular appendices of patients with cryptorchidism pressure to which these testes are exposed. show significant alterations in the estrogen and an- In conclusion, we did not find a difference drogen receptors (19,23). Patients with hydroceles in the incidence of testicular appendices in relation also present alteration in the expression of andro- to the testicular position in the patients with cryp- gen receptors and epithelial destruction in the testi- torchidism. There also was no increased incidence cular appendices (19). These alterations are argued of anatomical anomalies associated with the testes to be associated with increased hydrostatic pressure containing appendices. The testicular appendices in the scrotum of patients with hydroceles (19,23). showed a significant structural alteration in the pa-

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